Cellular and electrophysiological characterization of triadin knockout syndrome using induced pluripotent stem cell-derived cardiomyocytes.

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two unrelated TKOS patients and an unrelated control. CRISPR-Cas9 gene editing was used to insert homozygous TRDN-p.D18fs∗13 into a control line to generate a TKOS model (TRDN-/-). Western blot confirmed total knockout of triadin in patient-specific and TRDN-/- iPSC-CMs. iPSC-CMs from both patients revealed a prolonged action potential duration (APD) at 90% repolarization, and this was normalized by protein replacement of triadin. APD prolongation was confirmed in TRDN-/- iPSC-CMs. TRDN-/- iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of key calcium handling proteins, slow and decreased calcium release from the sarcoplasmic reticulum, and slow inactivation of the L-type calcium channel leading to frequent cellular arrhythmias, including early and delayed afterdepolarizations and APD alternans.

SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19.

BACKGROUND: SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. OBJECTIVE: To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca2+ indicator. RESULTS: Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P<0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P<0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large "tsunami"-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca2+ handling returned to normal. CONCLUSION: The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte's repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

Effect of pneumatic leg compression on post-induction hypotension in elderly patients undergoing robot-assisted laparoscopic prostatectomy: a double-blind randomised controlled trial.

Post-induction hypotension is common and associated with postoperative complications. We hypothesised that pneumatic leg compression reduces post-induction hypotension in elderly patients undergoing robot-assisted laparoscopic prostatectomy. In this double-blind randomised study, patients were allocated randomly to the pneumatic leg compression group (n = 50) or control (n = 50). In the intervention group, pneumatic leg compression was initiated before induction of anaesthesia. In the control group, pneumatic leg compression was initiated 20 min after anaesthesia induction. The primary outcome was the incidence of post-induction hypotension in these groups. Post-induction hypotension was defined as systolic blood pressure < 90 mmHg during the first 20 min after induction. Haemodynamic variables and area under the curve of post-induction systolic blood pressure over time were assessed. Complications associated with pneumatic leg compression were recorded, including: peripheral neuropathy; compartment syndrome; extensive bullae beneath the leg sleeves; and pulmonary thromboembolism. The incidence of post-induction hypotension decreased in the pneumatic leg compression group compared with that in the control group; 5 (10%) vs. 29 (58%), respectively, p < 0.001. In the pneumatic leg compression group, the lowest systolic, diastolic and mean blood pressures 20 min after induction of anaesthesia were significantly greater than the control group. Pneumatic leg compression resulted in an increased area under the curve of systolic blood pressure in the first 20 min after induction, p = 0.001. There were no pneumatic leg compression-related complications. Pneumatic leg compression reduced post-induction hypotension in elderly patients undergoing robot-assisted laparoscopic prostatectomy, suggesting that it is an effective and safe intervention to prevent post-induction hypotension among elderly patients undergoing general anaesthesia.

Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca2+ imaging assessed Ca2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca2+ sparking and Ca2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca2+ leak-triggered arrhythmias.

Acacetin, a Potent Transient Outward Current Blocker, May Be a Novel Therapeutic for KCND3-Encoded Kv4.3 Gain-of-Function-Associated J-Wave Syndromes.

BACKGROUND: The transient outward current (Ito) that mediates early (phase 1) repolarization is conducted by the KCND3-encoded Kv4.3 pore-forming α-subunit. KCND3 gain-of-function mutations have been reported previously as a pathogenic substrate for J wave syndromes (JWS), including the Brugada syndrome and early repolarization syndrome, as well as autopsy-negative sudden unexplained death (SUD). Acacetin, a natural flavone, is a potent Ito current blocker. Acacetin may be a novel therapeutic for KCND3-mediated J wave syndrome. METHODS: KCND3-V392I was identified in an 18-year-old male with J wave syndrome/early repolarization syndrome, and a history of cardiac arrest including ventricular tachycardia/ventricular fibrillation and atrial fibrillation/atrial flutter. Pathogenic KCND3 mutation was engineered by site-directed mutagenesis and co-expressed with wild-type KChIP2 in TSA201 cells. Gene-edited/variant-corrected isogenic control and patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the p. Val392Ile-KCND3-positive patient were generated. Ito currents and action potentials were recorded before and after treatment with Acacetin using the whole cell patch-clamp and multielectrode array technique. Western blot and immunocytochemistry were performed to investigate KCND3 expression. RESULTS: KCND3-V392I demonstrated a marked gain-of-function phenotype, increasing peak Ito current density by 92.2% (P<0.05 versus KCND3-WT). KCND3 expression was significantly increased in KCND3-V392I-derived iPSC-CMs (P<0.05 versus isogenic control). While KCND3-WT revealed an IC50 of 7.2±1.0 µmol/L for acacetin effect, 30 µmol/L acacetin dramatically inhibited KCND3-V392I peak Ito current density by 96.2% (P<0.05 versus before Acacetin). Ito was also increased by 60.9% in Kv4.3-V392I iPSC-CM (P<0.05 versus isogenic control iPSC-CM). Ten micromoles per liter acacetin, a concentration approaching its IC50 value, inhibited Ito by ≈50% in patient-derived iPSC-CMs and reduced the accentuated action potential notch displayed in KCND3-V392I-derived iPSC-CMs. CONCLUSIONS: This preclinical study provides pharmacological and functional evidence to suggest that Acacetin may be a novel therapeutic for patients with KCND3 gain-of-function-associated J wave syndrome by inhibiting Ito and abolishing the accentuated action potential notch in patient-derived iPSC-CMs.

Seasonal variability of ocean circulation near the Dotson Ice Shelf, Antarctica.

Recent rapid thinning of West Antarctic ice shelves are believed to be caused by intrusions of warm deep water that induce basal melting and seaward meltwater export. This study uses data from three bottom-mounted mooring arrays to show seasonal variability and local forcing for the currents moving into and out of the Dotson ice shelf cavity. A southward flow of warm, salty water had maximum current velocities along the eastern channel slope, while northward outflows of freshened ice shelf meltwater spread at intermediate depth above the western slope. The inflow correlated with the local ocean surface stress curl. At the western slope, meltwater outflows followed the warm influx along the eastern slope with a ~2-3 month delay. Ocean circulation near Dotson Ice Shelf, affected by sea ice distribution and wind, appears to significantly control the inflow of warm water and subsequent ice shelf melting on seasonal time-scales.

Patient-specific, re-engineered cardiomyocyte model confirms the circumstance-dependent arrhythmia risk associated with the African-specific common SCN5A polymorphism p.S1103Y: Implications for the increased sudden deaths observed in black individuals during the COVID-19 pandemic.

BACKGROUND: During the early stages of the coronavirus disease 2019 (COVID-19) pandemic, a marked increase in sudden cardiac death (SCD) was observed. The p.S1103Y-SCN5A common variant, which is present in ∼8% of individuals of African descent, may be a circumstance-dependent, SCD-predisposing, proarrhythmic polymorphism in the setting of hypoxia-induced acidosis or QT-prolonging drug use. OBJECTIVE: The purpose of this study was to ascertain the effects of acidosis and hydroxychloroquine (HCQ) on the action potential duration (APD) in a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of p.S1103Y-SCN5A. METHODS: iPSC-CMs were generated from a 14-year-old p.S1103Y-SCN5A-positive African American male. The patient's variant-corrected iPSC-CMs (isogenic control [IC]) were generated using CRISPR/Cas9 technology. FluoVolt voltage-sensitive dye was used to assess APD90 values in p.S1103Y-SCN5A iPSC-CMs compared to IC before and after an acidotic state (pH 6.9) or 24 hours of treatment with 10 µM HCQ. RESULTS: Under baseline conditions (pH 7.4), there was no difference in APD90 values of p.S1103Y-SCN5A vs IC iPSC-CMs (P = NS). In the setting of acidosis (pH 6.9), there was a significant increase in fold-change of APD90 in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). Similarly, with 24-hour 10 µM HCQ treatment, the fold-change of APD90 was significantly higher in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). CONCLUSION: Although the African-specific p.S1103Y-SCN5A common variant had no effect on APD90 under baseline conditions, the physiological stress of either acidosis or HCQ treatment significantly prolonged APD90 in patient-specific, re-engineered heart cells.

Development of a Patient-Specific p.D85N-Potassium Voltage-Gated Channel Subfamily E Member 1-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for Drug-Induced Long QT Syndrome.

BACKGROUND: Prior epidemiological studies demonstrated that the p.D85N-Potassium voltage-gated channel subfamily E member 1 (KCNE1) common variant reduces repolarization reserve and predisposes to drug-induced QT prolongation/torsades de pointes. We sought to develop a cellular model for drug-induced long QT syndrome using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM). METHODS: p.D85N-KCNE1 iPSCs were generated from a 23-year-old female with an exaggerated heart rate-corrected QT interval response to metoclopramide (ΔQTc of 160 ms). Clustered regularly interspaced short palindromic repeats-associated 9 technology was used to generate gene-corrected isogenic iPSCs. Field potential duration and action potential duration (APD) were measured from iPSC-CMs. RESULTS: At baseline, p.D85N-KCNE1 iPSC-CMs displayed significantly longer field potential duration (281±15 ms, n=13 versus 223±8.6 ms, n=14, P<0.01) and action potential duration at 90% repolarization (APD90; 579±22 ms, n=24 versus 465±33 ms, n=26, P<0.01) than isogenic-control iPSC-CMs. Dofetilide at a concentration of 2 nM increased significantly field potential duration (379±20 ms, n=13, P<0.01) and APD90 (666±11 ms, n=46, P<0.01) in p.D85N-KCNE1 iPSC-CMs but not in isogenic-control. The effect of dofetilide on APD90 (616±54 ms, n=7 versus 526±54 ms, n=10, P<0.05) was confirmed by Patch-clamp. Interestingly, treatment of p.D85N-KCNE1 iPSC-CMs with estrogen at a concentration of 1 nM exaggerated further dofetilide-induced APD90 prolongation (696±9 ms, n=81, P<0.01) and caused more early afterdepolarizations (11.7%) compared with isogenic control (APD90: 618±8 ms, n=115 and early afterdepolarizations: 2.6%, P<0.05). CONCLUSIONS: This iPSC-CM study provides further evidence that the p.D85N-KCNE1 common variant in combination with environmental factors such as QT prolonging drugs and female sex is proarrhythmic.

Suppression-Replacement KCNQ1 Gene Therapy for Type 1 Long QT Syndrome.

BACKGROUND: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1-encoded Kv7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. No current therapies target the molecular cause of LQT1. METHODS: A dual-component suppression-and-replacement (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 short hairpin RNA and a short hairpin RNA-immune KCNQ1 cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from 4 patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for 2 LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. RESULTS: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune KCNQ1 as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls. CONCLUSIONS: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, cotransfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression and replacement of KCNQ1. In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

Promise and Potential Peril With Lumacaftor for the Trafficking Defective Type 2 Long-QT Syndrome-Causative Variants, p.G604S, p.N633S, and p.R685P, Using Patient-Specific Re-Engineered Cardiomyocytes.

BACKGROUND: The KCNH2-encoded Kv11.1 hERG (human ether-a-go-go related gene) potassium channel is a critical regulator of cardiomyocyte action potential duration (APD). The majority of type 2 long-QT syndrome (LQT2) stems from trafficking defective KCNH2 mutations. Recently, Food and Drug Administration-approved cystic fibrosis protein trafficking chaperone, lumacaftor, has been proposed as novel therapy for LQT2. Here, we test the efficacy of lumacaftor treatment in patient-specific induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) derived from 2 patients with known LQT2 trafficking defective mutations and a patient with novel KCNH2 variant, p.R685P. METHODS: Patient-specific iPSC-CM models of KCNH2-G604S, KCNH2-N633S, and KCNH2-R685P were generated from 3 unrelated patients diagnosed with severe LQT2 (rate-corrected QT>500 ms). Lumacaftor efficacy was also tested by ANEPPS, FluoVolt, and ArcLight voltage dye-based APD90 measurements. RESULTS: All 3 mutations were hERG trafficking defective in iPSC-CMs. While lumacaftor treatment failed to rescue the hERG trafficking defect in TSA201 cells, lumacaftor rescued channel trafficking for all mutations in the iPSC-CM model. All 3 mutations conferred a prolonged APD90 compared with control. While lumacaftor treatment rescued the phenotype of KCNH2-N633S and KCNH2-R685P, lumacaftor paradoxically prolonged the APD90 in KCNH2-G604S iPSC-CMs. Lumacaftor-mediated APD90 rescue was affected by rapidly activating delayed rectifier K+ current blocker consistent with the increase of rapidly activating delayed rectifier K+ current by lumacaftor is the underlying mechanism of the LQT2 rescue. CONCLUSIONS: While lumacaftor is an effective hERG channel trafficking chaperone and may be therapeutic for LQT2, we urge caution. Without understanding the functionality of the mutant channel to be rescued, lumacaftor therapy could be harmful.

Molecular characterization of the calcium release channel deficiency syndrome.

We identified a potentially novel homozygous duplication involving the promoter region and exons 1-4 of the gene encoding type 2 cardiac ryanodine receptor (RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.

Identification of a Novel Homozygous Multi-Exon Duplication in RYR2 Among Children With Exertion-Related Unexplained Sudden Deaths in the Amish Community.

Importance: The exome molecular autopsy may elucidate a pathogenic substrate for sudden unexplained death. Objective: To investigate the underlying cause of multiple sudden deaths in young individuals and sudden cardiac arrests that occurred in 2 large Amish families. Design, Setting, and Participants: Two large extended Amish families with multiple sudden deaths in young individuals and sudden cardiac arrests were included in the study. A recessive inheritance pattern was suggested based on an extended family history of sudden deaths in young individuals and sudden cardiac arrests, despite unaffected parents. A family with exercise-associated sudden deaths in young individuals occurring in 4 siblings was referred for postmortem genetic testing using an exome molecular autopsy. Copy number variant (CNV) analysis was performed on exome data using PatternCNV. Chromosomal microarray validated the CNV identified. The nucleotide break points of the CNV were determined by mate-pair sequencing. Samples were collected for this study between November 2004 and June 2019. Main Outcomes and Measures: The identification of an underlying genetic cause for sudden deaths in young individuals and sudden cardiac arrests consistent with the recessive inheritance pattern observed in the families. Results: A homozygous duplication, involving approximately 26 000 base pairs of intergenic sequence, RYR2's 5'UTR/promoter region, and exons 1 through 4 of RYR2, was identified in all 4 siblings of a family. Multiple distantly related relatives experiencing exertion-related sudden cardiac arrest also had the identical RYR2 homozygous duplication. A second, unrelated family with multiple exertion-related sudden deaths and sudden cardiac arrests in young individuals, with the same homozygous duplication, was identified. Several living, homozygous duplication-positive symptomatic patients from both families had nondiagnostic cardiologic testing, with only occasional ventricular ectopy occurring during exercise stress tests. Conclusions and Relevance: In this analysis, we identified a novel, highly penetrant, homozygous multiexon duplication in RYR2 among Amish youths with exertion-related sudden death and sudden cardiac arrest but without an overt phenotype that is distinct from RYR2-mediated catecholaminergic polymorphic ventricular tachycardia. Considering that no cardiac tests reliably identify at-risk individuals and given the high rate of consanguinity in Amish families, identification of unaffected heterozygous carriers may provide potentially lifesaving premarital counseling and reproductive planning.

FeSe quantum dots for in vivo multiphoton biomedical imaging.

An immense demand in biomedical imaging is to develop efficient photoluminescent probes with high biocompatibility and quantum yield, as well as multiphoton absorption performance to improve penetration depth and spatial resolution. Here, iron selenide (FeSe) quantum dots (QDs) are reported to meet these criteria. The synthesized QDs exhibit two- and three-photon excitation property at 800- and 1080-nm wavelengths and high quantum yield (ca. 40%), which are suitable for second-window imaging. To verify their biosuitability, poly(ethylene glycol)-conjugated QDs were linked with human epidermal growth factor receptor 2 (HER2) antibodies for in vitro/in vivo two-photon imaging in HER2-overexpressed MCF7 cells and a xenograft breast tumor model in mice. Imaging was successfully carried out at a depth of up to 500 µm from the skin using a nonlinear femtosecond laser at an excitation wavelength of 800 nm. These findings may open up a way to apply biocompatible FeSe QDs to multiphoton cancer imaging.

MRAS Variants Cause Cardiomyocyte Hypertrophy in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Additional Evidence for MRAS as a Definitive Noonan Syndrome-Susceptibility Gene.

BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.

Characterization of the CACNA1C-R518C Missense Mutation in the Pathobiology of Long-QT Syndrome Using Human Induced Pluripotent Stem Cell Cardiomyocytes Shows Action Potential Prolongation and L-Type Calcium Channel Perturbation.

BACKGROUND: The CACNA1C-encoded cardiac L-type calcium channel (Cav1.2) is essential for cardiocyte action potential duration (APD). We previously reported the CACNA1C-p.R518C variant associated with prolonged QT intervals, cardiomyopathy, and sudden cardiac death in several pedigrees. METHODS: To characterize a patient-derived human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) CACNA1C-p.R518C model, CACNA1C-p.R518C hiPSC-CMs were generated from a 13-year-old man (QTc, >480 ms) with a family history of sudden cardiac death. An isogenic hiPSC-CM gene-corrected control was created using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9). APD and calcium handling were assessed by live cell imaging with Arclight voltage and Fluo-4 calcium indicators, respectively. The APD and L-type calcium channel biophysical properties were further assessed by whole-cell patch clamp technique. RESULTS: The Bazett formula-corrected, Arclight-measured APD90 of CACNA1C-p.R518C hiPSC-CMs was significantly longer (622±11 ms; n=92) than the isogenic control hiPSC-CMs (453±5 ms; n=62; P<0.0001). Patch clamp assessment of CACNA1C-p.R518C hiPSC-CMs paced at 1 Hz confirmed a prolonged APD90 (689±29 ms; n=10) compared with the patient's isogenic control hiPSC-CMs (434±30 ms; n=8; P<0.05). Fluo-4-measured calcium transient decay time suggested calcium mishandling in CACNA1C-p.R518C. Patch clamp analysis revealed increased L-type calcium channel window current, slow decay time at various voltages, and increased late calcium current for CACNA1C-p.R518C hiPSC-CMs when compared with isogenic control hiPSC-CMs. CONCLUSIONS: Using patient-specific hiPSC-CM mutant and isogenic control lines, we demonstrate that the CACNA1C-p.R518C variant is the self-sufficient, monogenetic substrate for the patient's long-QT syndrome phenotype. These data further bolster the conclusion that CACNA1C is a bona fide, definite evidence long-QT syndrome susceptibility gene.

Systemic lupus erythematosus is a risk factor for cancer: a nationwide population-based study in Korea.

OBJECTIVE: Specific differences in cancer risk have been observed between systemic lupus erythematosus patients and the general population. Although meta-analyses have estimated cancer incidence in systemic lupus erythematosus patients, results have been inconclusive. Hence, we aimed to assess malignancy risk in systemic lupus erythematosus patients, compared to the risk in the general population. METHODS: Systemic lupus erythematosus patients ( n = 21,016; mean age 41.67 ± 13.14 years; female 90.22%) were selected from the Korean National Health Insurance Service database between 2008 and 2014. Age- and sex-matched controls were randomly sampled in a 5:1 ratio ( n = 105,080). RESULTS: During the 7 years of follow up, malignancy was detected in 763 (3.63%) systemic lupus erythematosus patients and 2667 (2.54%) controls. Systemic lupus erythematosus patients had a higher risk of malignancy than controls (odds ratio 1.44; 95% confidence interval 1.327-1.559), after multivariate adjustment. Systemic lupus erythematosus patients had a higher odds ratio for developing cervical, thyroid, ovarian, and oral cancer, as well as lymphoma, leukemia, and multiple myeloma than controls. Based on subgroup analysis, male systemic lupus erythematosus patients and patients younger than 40 years showed the highest lymphoma risk. CONCLUSIONS: Systemic lupus erythematosus might be an independent risk factor for cancer. Therefore, the importance of cancer screening programs should be emphasized in systemic lupus erythematosus patients. Our study is the first large nationwide cohort study for evaluating the risk of cancer in systemic lupus erythematosus patients.

Surgical Therapy of Peri-Implantitis with Local Minocycline: A 6-Month Randomized Controlled Clinical Trial.

The purpose of this study was to determine the clinical, microbial, and radiographic effects of local minocycline combined with surgical treatment of peri-implantitis. Fifty patients with peri-implantitis were recruited, and surgical treatment with the local application of either minocycline or placebo ointment was performed. The application of minocycline was repeated with supragingival debridement at 1, 3, and 6 mo postoperatively. Plaque index, gingival index (GI), probing pocket depth (PPD), and bleeding/suppuration on probing were measured at baseline and 1-, 3-, and 6-mo evaluations. The change in supporting bone level (SBL) measured with cone beam computed tomography was analyzed between baseline and 6 mo. Microbial analysis was performed with real-time polymerase chain reaction. Both groups exhibited improvements in clinical and radiographic measurements after surgical treatment. There was a significant difference in the changes of mean PPD between the test and control groups (2.68 ± 1.73 and 1.55 ± 1.86 mm, respectively, P = 0.039). The changes of mean GI and SBL differed significantly between the groups (ΔGI: 0.83 ± 0.60 and 0.40 ± 0.68; ΔSBL: 0.72 ± 0.56 and 0.31 ± 0.49 mm, respectively, P = 0.026 and 0.014). Treatment success rates (defined as PPD <5 mm, absence of bleeding/suppuration on probing, and no further bone loss) were 66.7% and 36.3% in the test and control groups, respectively. The count of red complex bacteria tended to decrease in both groups until 6 mo; however, no significant intergroup difference was found. None of the patients in the test group carried Porphyromonas gingivalis or Tannerella forsythia at 6 mo. These findings indicate that the repeated local delivery of minocycline combined with surgical treatment provides significant benefits in terms of clinical parameters and radiographic bone fill, with a higher treatment success rate in the short healing period (cris.nih.go.kr KCT0002844).

Survival outcome differences based on treatments used and knowledge of the primary tumour site for patients with cancer of unknown and known primary in Ontario.

Introduction: Patients with cancer of unknown primary (cup) have pathologically confirmed metastatic tumours with unidentifiable primary tumours. Currently, very little is known about the relationship between the treatment of patients with cup and their survival outcomes. Thus, we compared oncologic treatment and survival outcomes for patients in Ontario with cup against those for a cohort of patients with metastatic cancer of known primary site. Methods: Using the Ontario Cancer Registry and the Same-Day Surgery and Discharge Abstract databases maintained by the Canadian Institute for Health Information, we identified all Ontario patients diagnosed with metastatic cancer between 1 January 2000 and 31 December 2005. Ontario Health Insurance Plan treatment records were linked to identify codes for surgery, chemotherapy, or therapeutic radiation related to oncology. Multivariable Cox regression models were constructed, adjusting for histology, age, sex, and comorbidities. Results: In 45,347 patients (96.3%), the primary tumour site was identifiable, and in 1743 patients (3.7%), cup was diagnosed. Among the main tumour sites, cup ranked as the 6th largest. The mean Charlson score was significantly higher (p < 0.0001) in patients with cup (1.88) than in those with a known primary (1.42). Overall median survival was 1.9 months for patients with cup compared with 11.9 months for all patients with a known-primary cancer. Receipt of treatment was more likely for patients with a known primary site (n= 35,012, 77.2%) than for those with cup (n = 891, 51.1%). Among patients with a known primary site, median survival was significantly higher for treated than for untreated patients (19.0 months vs. 2.2 months, p < 0.0001). Among patients with cup, median survival was also higher for treated than for untreated patients (3.6 months vs. 1.1 months, p < 0.0001). Conclusions: In Ontario, patients with cup experience significantly lower survival than do patients with metastatic cancer of a known primary site. Treatment is associated with significantly increased survival both for patients with cup and for those with metastatic cancer of a known primary site.

Systemic lupus erythematosus is a risk factor for cardiovascular disease: a nationwide, population-based study in Korea.

OBJECTIVE: To investigate the incidence and clinical significance of cardiovascular disease in systemic lupus erythematosus patients. METHODS: We included systemic lupus erythematosus patients ( n = 18,575) without previous cardiovascular disease and age- and sex-matched individuals without systemic lupus erythematosus (controls; n = 92,875) from the Korean National Health Insurance Service database (2008-2014). Both cohorts were followed up for incident cardiovascular disease and death until 2015. RESULTS: During follow up, myocardial infarction occurred in 203 systemic lupus erythematosus patients and 325 controls (incidence rate: 1.76 and 0.56 per 1000 person-years, respectively), stroke occurred in 289 patients and 403 controls (incidence rate: 2.51 and 0.70 per 1000 person-years, respectively), heart failure occurred in 358 patients and 354 controls (incidence rate 3.11 and 0.61 per 1000 person-years, respectively), and death occurred in 744 patients and 948 controls (incidence rate 6.54 and 1.64 per 1000 person-years, respectively). Patients with systemic lupus erythematosus had higher risks for myocardial infarction (hazard ratio: 2.74, 95% confidence interval: 2.28-3.37), stroke (hazard ratio: 3.31, 95% confidence interval: 2.84-3.86), heart failure (hazard ratio: 4.60, 95% confidence interval: 3.96-5.35), and cardiac death (hazard ratio: 3.98, 95% confidence interval: 3.61-4.39). CONCLUSIONS: Here, systemic lupus erythematosus was an independent risk factor for cardiovascular disease, thus cardiac assessment and management are critical in systemic lupus erythematosus patients.

Two-dimensional plasmonic grating for increased quantum efficiency in midwave infrared nBn detectors with thin absorbers.

We demonstrate a strategy for increasing the operating temperatures of nBn midwave infrared (MWIR) focal plane arrays, based on the use of two-dimensional plasmonic gratings to enhance the quantum efficiency (QE) of structures with very thin absorbers. Reducing the absorber volume correspondingly reduces the dark current in a diffusion-limited photodiode, while light trapping mediated by the plasmonic grating increases the net absorbance to maintain high QE. The plasmonically enhanced nBn MWIR sensors with absorber thicknesses of only 0.5 µm exhibit peak internal QEs as high as 57%, which enables a 5-fold reduction in dark current. Numerical simulations indicate the potential for further improvement.